2,3,7,8-tetrachloro-dibenzo-p-dioxin ("TCDD") is one of the most potent small molecule toxins known to man. It was a contaminant in the Vietnam defoliant known as "Agent Orange". The widespread dispersion of dioxins such as TCDD in our environment, their resistance to degradation, their lipophilicity (and hence accumulation in the food chain), and their toxic potency have all led to concern about the hazards they pose. Considerable effort has therefore been devoted to monitoring the concentration of these compounds.
Testing for trace concentrations of these compounds in environmental and biological samples has been made possible through recent advances in chromatographic and mass spectral technologies. These methods usually involve varying degrees of sample preparation, separation of isomers by liquid or gas chromatography, and identification and quantification of related compounds by mass spectrometry. Despite the sensitivity of mass spectrometer methods, their use is limited by cost and the availability of instrumentation.
Bioassays, such as the formation of pericardial edema in newborn chicks or production of chloracne in rabbit ears, played a prominent early role in the identification and monitoring of chlorinated dibenzo-p-dioxins. Bioassays using cell cultures (e.g., induction of aryl hydrocarbon hydroxylase activity in rat hepatoma cells o keratinization in XB/3T3 cells, have achieved appreciable sensitivity (approximately 10 pg), but have not gained widespread use. The primary reasons such methods are not widely used are that they are time consuming, costly, and suffer from the variability inherent in using whole cells.
Radioimmunoassays (using antibodies and certain radioactively labelled ligands) to detect dioxins have been developed with sensitivities of approximately 25 pg. See generally P. Albro et al., 50 Tox. Appl. Pharm. 137-146 (1979). The limited use of such RIA techniques to date with dioxins may be attributed to 1) the need to characterize each antisera for its reactivity towards a large number of isomers and interfering compounds; 2) difficulties in the solubility of antigen; and 3) lack of and/or cost of optimal radioligands. In this regard, dioxins are too small to efficiently form antibodies until they are altered with complex linking structures.
It has also been known that dioxins exert their biological effects via stereospecific binding to a soluble protein, the aryl hydrocarbon hydroxylase receptor ("Ah receptor"). Attempts have therefore been made to competitively bind Ah receptor to C.sup.14 and .sup.3 H radioactive variants of dioxins as an assay technique. However, such compounds have proven to have unacceptably low specific activity for commercial assays or other problems. Radioactive ligands having appropriate activity and appropriate stability, specificity, and solubility, vis a vis Ah receptor have not to date been developed. Thus, it can be seen that the need exists for an improved assay for dioxins.